The klotho gene is known to play a role in suppressing several different aging phenotypes, based on studies in the klotho mutant mouse. Mice homozygous for defects in the klotho gene exhibit a syndrome that closely resembles human aging, including atherosclerosis, osteoporosis, emphysema, infertility and skin atrophy. We have looked for sequence variation in the klotho gene in a paenl of well-characterized mouse strains from the Jackson Laboratory. Each exon of the klotho gene was sequenced in each of these 20 strains.This work is now being submitted to the journal Genomics. Specifically, we have found: 1. No variation was found in any of the 16 laboratory derived inbred stains in the panel. 2.Among the 4 wild-derived inbred strains, 45 variants were found, including 43 single nucleotide substitutions, one deletion and one insertion. Of the nucleotide substitutions, six resulted in amino acid substitutions. 3.Real-time RT-PCR analysis of klotho gene expression in the wild-derived strains has shown a higher level of gene expression in SPRET/Ei than in the other wild-derived or laboratory-derived strains. 4. The ratio of membrane form to secreted form of klotho mRNA is higher in the three wild-derived strains with amino acid substitutions that in the control strains. Our finding that the klotho mRNA in the wild-derived species SPRET/Ei is expressed at approximately twice the level of laboratory derived strains and has four amino acid changes is intriguing in light of several phenotypic differences between SPRET/Ei and laboratory derived strains that may be related to klotho expression. These phenotypic differences include a long life-span, exceptional hearing, low total cholesterol and high HDL levels. Interestingly, MOLF/Ei, which exhibited the lowest klotho expression levels of the wild-derived strains, had the highest total cholesterol levels and lowest percent HDL of the 43 strains tested in the mouse phenome project. We hypothesize that klotho mRNA alterations and increased expression levels in SPRET/Ei may provide protection against age-related diseases such as hearing loss and coronary artery disease. It is also possible that specific klotho variants in mice may be associated with increased longevity, as has been seen in humans. Our IRB-approved protocol to look for functional variants of the Klotho gene among participants in the Baltimore Longitudinal Study on Aging was expanded this year to include the InChianti population as well. We have obtained DNA from BLSA participants with atherosclerosis, type II diabetes, osteoporosis and osteoarthritis and begun to type DNA from patients with coronary artery disease, stroke and atherosclerosis, as well as healthy controls, for the presence of the KL-VS Klotho allele. Preliminary analysis of this data suggests that there is no difference in the frequency of the KL-VS allele between participants affected with athersclerotic disease and those without. Further genotyping and data analysis will continue in the coming year. We have also completed the process of DNA isolation from 1200 samples in the InChianti population. Over the coming months we will be genotyping the entire InChianti population for the KL-VS allele, and looking for new variants in the Klotho gene among this Italian population. Data analysis will involve a search for correlations between specific allelic variants and phenotypes examined in the InChianti study. An abstract describing the initial analysis of atherosclerosis and Klotho allele status in the BLSA population was sent to the American Society of Human Genetics for presentation at the 2003 meeting in October: Search for association with Klotho allele variation and atherosclerosis in the Baltimore Longitudinal Study on Aging. A. Bektas, D. Taub, S. Najjar, D. Muller, L. Ferrucci, C. A. Francomano.